The major objective of this proposal is to develop methods for the site specific insertion of one or more amino acids analogues into proteins in eubacteria and in eukaryotes. The amino acid analogues to be used could include those that are photoactivatable, those that are fluorescent, those that carry reactive side chains such as keto and azido groups, heavy atoms such as iodine, spectroscopic probes and those that mimic phospho-amino acids. Besides providing a method for the production of proteins with novel chemical and biological properties, proteins carrying such amino acid analogues will have wide applications in biology including studies on the folding, structure, stability and function of proteins, protein-protein interactions, protein localization and protein dynamics in vivo, crystallographic structure analyses and signal transduction. Two approaches are proposed. The first consists of the use of an amber suppressor transfer RNA (tRNA) aminoacylated in vivo with an amino acid analogue with the help of a mutant aminoacyl-tRNA synthetase, to insert the amino acid analogue at a specific site in a protein. A second, more general, approach is based on the finding in the investigator's laboratory that suppressor tRNAs, including those that are aminoacylated, can be imported into mammalian cells. This finding opens up the possibility of importing suppressor tRNAs aminoacylated in vitro with a variety of amino acid analogues for the site specific insertion of one or more amino acid analogues into proteins. The specific aims are: (1)To isolate mutants of (i) yeast tyrosyl-tRNA synthetase, which aminoacylate amber suppressor tRNA with iodotyrosine, p-trifluoromethyl-diazirinylphenylalanine or p-azido phenylalanine and (ii) E. coli glutaminyl-tRNA synthetase, which aminoacylate amber suppressor tRNA with 2-amino 5-keto hexanoic acid, an analogue of glutamine in which the side chain -CH2CH2CONH2 is replaced by - CH2CH2COCH3. (2) To identify, produce and purify amber and ochre suppressor tRNAs, which are not aminoacylated by mammalian aminoacyl-tRNA synthetases and which are highly active in suppression in mammalian cells. (3) To optimize the conditions for import of suppressor tRNAs aminoacylated with amino acid analogues into mammalian cells and to use them for site specific insertion into proteins.